Hepatitis B virus (HBV) causes acute or chronic hepatitis, which may progress to liver cirrhosis and liver cancer. HBV is a DNA virus which replicates via an RNA intermediate and utilizes reverse transcription in its replication strategy [Summers and Mason, Cell 29; 403-415, 1982]. HBV DNA polymerase is responsible for the reverse transcription and has been considered the main target for anti-HBV intervention. Many nucleoside or nucleotides analogs have been discovered to be effective anti-viral agents. Examples of nucleoside analogs which have been tested are penciclovir and its oral form (FCV) [Vere Hodge, Antiviral Chem Chemother. 4: 67-84, 1993; Kruger et al., Hepatology 22: 219A, 1994; Main et al., J. Viral Hepatitis 3: 211-215, 1996], and Lamivudine[(−)-B-2′-deoxy-3′-thiacytidine]; (3TC or LMV) [Severini et al., Antimicrobial Agents Chemother. 39: 430-435, 1995; Dienstag et al., New England J Med 333: 1657-1661, 1995]. New nucleoside or nucleotide analogs that have progressed to clinical trials or are approved for HBV by FDA include Emtricitabine (FTC), Clevudine (L-FMAU), Entecavir (BMS-200, 475; ETV), diaminopurine dioxolane (DAPD), adefovir dipivoxil. (9-(2-((bis((pivaloyloxy)methoxy)phosphinyl)methoxy)-ethyl)adenine). Additionally, for a number of years interferon alpha also has been widely used for the treatment of chronic HBV infection.
Although these agents are highly effective in inhibiting HBV DNA synthesis, resistant mutants of HBV have emerged during long term nucleoside or nucleotide antiviral chemotherapy. Sustained responses to HBV treatment—as evidenced by a decrease of HBV DNA in serum and by anti-HBe or HBs seroconversion—has been observed only in a relatively small patient population.
For example, for several years interferon alpha has been widely used for the treatment of chronic HBV infection. However, interferon is effective only in certain subpopulations of chronic hepatitis B patients, and even in such patients it is poorly tolerated. Similarly, lamivudine (3′-thia-2′,3′-dideoxycytidine), a particularly strong inhibitor of HBV replication, is used to treat HBV infection. However, resistance to lamivudine is increasingly common and has limited its efficacy in a high proportion of patients. The most recently-approved treatment for HBV is adefovir dipivoxil (9-(2-((bis((pivaloyloxy)methoxy)phosphinyl)methoxy)ethyl)adenine). Although this nucleoside analog is active against the lamivudine-resistant viruses, its sustained viral response rate is poor (below 20%), and its maximum tolerated dose and treatment duration are often limited by nephrotoxicity.
More recent developments in HBV research have led to clinical trials for several compounds with promising antiviral activity. For example, certain nucleoside analogs have been reported to exhibit significant anti-HBV activity (e.g., 2′-fluoro-5-methyl-beta-L-arabinofuranosyluracil (Bukwang) and 2′-deoxy-5-fluoro-3′-thiacytidine (Gilead); 2′-deoxy-L-thymidine and 2′-deoxy-L-cytidine (both Idenix)). Similarly, carbocyclic nucleoside analogs (6H-purin-6-one, 2-amino-1,9-dihydro-9-((1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl) monohydrate (Bristol-Myers Squibb), as well as acyclic nucleoside analogs with liver targeting properties (Remofovir; Ribapharm), were reported as having anti-HBV activity in clinical trials.).
However, while most of the recently discovered drugs with anti-HBV activity exhibited promising in vitro antiviral activity, low response rates and the emergence of resistance limit the efficacy of these clinical candidates. Therefore, although various compositions and methods for HBV treatment are known in the art, there is still a need to provide new and improved compositions and methods for treatment of HBV infections in human patients.
Thus, in light of the limited efficacy, resistance profiles, and toxicity of current anti-HBV drugs, there is a strong need for novel anti-HBV drugs that are more effective and less toxic and that exhibit a different resistance profile.
Therefore, it is an object of the present invention to provide a compounds and methods for the treatment of HBV infection. Such compounds and methods also have potential for the treatment of other conditions associated with dysregulated protein kinase activity, such as inflammation and neoplastic disease.
The NF-κB pathway plays a complex role in an antiviral immune response. Nuclear factor-κB (NF-κB) is a ubiquitously expressed transcription factor that is essential to the regulation of such cellular functions as apoptosis, proliferation, and differentiation [Ghosh et al., Annu. Rev. Immunol. 16:225, 1998]. NF-κB accomplishes this regulation by coordinating the expression of genes responsible for protecting an organism after physical, chemical, and/or microbial damage. Thus, NF-κB has an inherent role in the induction of an immune response and concomitant inflammation. [Baeuerle and Baltimore. Cell 87:13, 1996]. The activity of NF-κB can be modulated by viral proteins. (Bose et al., PNAS 100, 2003; Purcell et al., Am. J. Physiol. Gastrointest. Liver Physiol. 280, 2001). Such effects can be both interferon-dependent and interferon-independent (Pfeffer et al., J. Biol. Chem. 279:30, 31304-31311, 2004).
The NF-κB family of transcription factors includes a set of structurally related and evolutionarily conserved DNA binding proteins (Baldwin, Annu. Rev. Immunol. 14:649, 1996). NF-κB contains a nuclear localization sequence (NLS) that directs the protein to the nucleus to carry out its role in genetic regulation. However, under normal conditions NF-κB is sequestered in the cytoplasm because the NLS is masked by tightly bound inhibitory proteins; these inhibitors of NF-κB are known as IκB (Beg and Baldwin, Genes Dev. 7:2064, 1993; Thompson et al., Cell 80:573, 1995; Whiteside et al., EMBO J. 16:1413, 1997). Activators of NF-κB act by inactivating IκB, via the mechanisms of phosphorylation, ubiquitination, and degradation. Thus, the elimination of IκB exposes the NLS allowing NF-κB to translocate to the nucleus to activate specific target genes.
The signal responsible for inactivation of IκB is typically a cellular response to an extracellular stimulus (Tumor Necrosis Factor α (TNFα), Interleukin-1β (IL-1β), lipopolysaccharide (LPS)) or to chemical and physical stress. The signal originates at a cell surface receptor, such as the TNF-receptor or IL-1 receptor; the signal is internalized and transduced through the cell via a cascade of phosphorylation events. Each receptor binds unique adapter molecules specific to the receptor and stimulus and in turn activates downstream kinases including NF-κB interacting kinase (NIK), MAPK/extracellular signal-regulated kinase kinase-1 (MEKK-1), and IκB kinases α and β (IKKα/β). IKKβ is responsible for liberating NF-κB by phosphorylating the inhibitory subunit IκBα. Phosphorylation of IκBα by IKKO triggers ubiquitin ligase (Skp1/Cul 1/F-box protein FWD1) to ubiquitinate IκBα and target it for degradation via the 26S proteasome [Yaron et al., Nature 396:590, 1998; Winston et al., Genes Dev. 13:270, 1999; Spencer et al., Genes Dev. 13:284, 1999].
The IκB kinases (TKKα and IKKβ) are serine-threonine protein kinases. They belong to a large multi-protein complex, called the “signalsome” [Mercurio et al., Science 278:860, 1997; Woronicz et al., Science 278:866, 1997; Zandi et al., 91:243, 1997]. The signalsome is the machinery responsible for transducing the stimulus that results in NF-κB activation. The genes that encode the signalsome components have been cloned, expressed, and reconstituted in vitro to demonstrate activation of NF-κB via IκB phosphorylation [Régnier et al., Cell 90:373, 1997; DiDonato et al., Nature 388:548, 1997; Zandi et al., Cell 91:243, 1997; Woronicz et al., Science 278: 866, 1997; Mercurio et al., Science 278:860, 1997; Cohen et al., Nature 395:292, 1998]. IKK family members share homologous amino-terminal kinase domains that are activated by NIK. In turn, IKK specifically phosphorylates IκBα and IκBβ on regulatory serine residues. Genetic studies with IKK knock-out mice point to an essential role for IKKβ in transmission of inflammatory signals, whereas IKKα is involved in developmental processes requiring NF-κB activation [Takeda et al., Science 284:313, 1999; Hu et al., Science 284:316, 1999; Li et al., Science 284:321, 1999]. Embryonic fibroblasts isolated from IKKβ-deficient mice show defects in TNFα- and IL-1-induced degradation of IκB. Furthermore, inhibition of pro-inflammatory cytokine-induced IκB degradation is not observed in cells derived from IKKα-deficient mice, suggesting that IKKβ controls the NF-κB activation rather than IKKα [Takeda et al., Science 284:313, 1999]. Moreover, a catalytically inactive mutant of IKKβ has been shown to inhibit inflammation via activation of NF-κB through TNFα, IL-1β, LPS, and anti-CD3/anti-CD28 stimulation [O'Connell et al., J. Biol. Chem. 273:30410, 1998; Woronicz et al., Science 278:866, 1997; Zandi et al., Cell 91:243, 1997.]. Thus, IKKβ is considered by the inventors to be a validated target for therapeutic interference in a variety of pathological situations, including chronic inflammatory and autoimmune diseases, viral infection, and cancer.
Some inhibitors of IKKβ have previously been reported. WO 03/103661, WO 01/58890, and WO 03/037886 describe substituted thienopyridines and heteroaromatic carboxamide derivatives as inhibitors of IKKβ. WO 01/68648 describes substituted β-carbolines having IKKβ inhibiting activity. Substituted indoles with IKKβ inhibitory activity are reported in WO 01/30774, and substituted benzimidazoles with NK-κB inhibitory activity are described in WO 01/00610. Recently, a number of imidazoloquinoxalines and related compounds have been reported to have IKK-inhibiting activity and to be useful in treating arthritis, transplant rejection, inflammatory bowel disease, and pulmonary inflammation disease in U.S. Pub. No. 2003/0022898. Additionally, aspirin and other salicylates have been reported to bind to and inhibit IKKβ (M. Yin et al., Nature, 1998, 396, 77).
Substituted thienopyridines that inhibit cell adhesion are reported in U.S. 2001/0020030 and in A. O. Stewart et al., J. Med. Chem., 2001, 44, 988. Thienopyridines with activity as antagonists of gonadotropin releasing hormone are reported in U.S. Pat. No. 6,313,301. Substituted thienopyridines described as telomerase inhibitors are disclosed in U.S. Pat. No. 5,656,638.